Towards unidirectional reconstitution of membrane proteins into liposomes

Abstract

Functional studies of membrane proteins (MP) in their native environment (the cellular membrane) is challenging due to the complexity of the native membranes and measurement interferences from other membrane constituents or reactions. Therefore, MPs are often purified and integrated into systems which mimic their natural environment in a membrane as e.g. liposomes or nanodiscs, a process called reconstitution, allowing to investigate its function and structural aspects without any disturbing background. The most important problem during MP-reconstitution is the often random orientation of the MP in the liposomal membrane after reconstitution. For functional studies of the MP of interest and quantitative analysis of its properties, unidirectional orientation in the liposomal membrane is required. Previous work of other groups, where MPs have been reconstituted and studied, did not include a final and universal approach (Rigaud JL, Levy D. (2003) Methods Enzymol. 372:65-86.; Tunuguntla R et al. (2013) Biophys. J. 105(6):1388-96.) and procedures have to be individually optimized for an enrichment of enzyme orientation. In most cases, however, orientation cannot be influenced and is thought to depend on the three-dimensional structure of the protein (Tunuguntla R et al. (2013) Biophys. J. 105(6):1388-96; Nordlund G et al. (2014) Nat. Commun. 5:4303.). We are currently developing and establishing a universal method to force unidirectional reconstitution of MPs by the aid of a molecular unit that can be attached to every protein. Our method is based on the observation that the F1F0 ATP synthase with its large hydrophilic head piece (F1 part) orients highly uniformly when embedded in preformed vesicles. We try to mimic this situation with a bioengineering approach that should be applicable to a wide variety of proteins. Once such a method is fully established, the interplay of two or more membrane proteins can be investigated more quantitatively.